Unlocking the potential of ancient hexaploid Indian dwarf wheat, Tritium sphaerococcum for grain quality improvement

Wild and ancient wheat are considered to be a rich source of nutrients and better stress tolerant, hence being re-considered for mainstreaming its cultivation by the farmers and bringing it back to the food basket. In the present study, thirty-four diverse accessions of Indian dwarf wheat, Triticum sphaerococcum conserved in the Indian National Genebank were evaluated for thirteen-grain quality parameters namely thousand-grain weight (TGW), hectolitre weight (HW), sedimentation value (Sed), grain hardness index (HI), protein (Pro), albumin (Alb), globulin (Glo), gliadin (Gli), glutenin (Glu), gluten, lysine (Lys), Fe2+ and Zn2+ content, and four antioxidant enzymes activities. Substantial variations were recorded for studied traits. TGW, HW, Sed, HI, Pro, Alb, Glo, Gli, Glu, Gluten, Lys, Fe2+, and Zn2+ varied from 26.50–45.55 g, 70.50–86.00 kg/hl, 24.00–38.00 ml, 40.49–104.90, 15.34–19.35%, 17.60–40.31 mg/g, 10.75–16.56 mg/g, 26.35–44.94 mg/g, 24.47–39.56 mg/g, 55.33–75.06 mg/g, 0.04–0.29%, 42.72–90.72 ppm, and 11.45–25.70 ppm, respectively. Among antioxidants, peroxidase (POX), catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD) activity ranged from 0.06–0.60 unit/ml, 0.02–0.61 unit/ml, 0.11–2.26 unit/ml, and 0.14–0.97 unit/ml, respectively. Hardness Index was positively associated with Pro and Zn2+ content whereas Lys was negatively associated with gluten content. Likewise, gluten and Fe2+ content had a positive association with the major protein fraction i.e., Gli and Glu. Hierarchical cluster analysis grouped 34 accessions into four clusters and the major group had nine indigenous and eight exotic accessions. We also validated high GPC accessions and EC182958 (17.16%), EC187176 and EC182945 (16.16%), EC613057 (15.79%), IC634028 (15.72%) and IC533826 (15.01%) were confirmed with more than 15% GPC. Also, superior trait-specific accessions namely, EC187167, IC534021, EC613055, EC180066, and EC182959 for low gluten content and IC384530, EC313761, EC180063, IC397363, EC10494 for high iron content (>76.51) were identified that may be used in wheat quality improvement for nutritional security of mankind.


INTRODUCTION
Worldwide, wheat is the most important cereal crop, contributing 55% of total carbohydrates, 21% of the total proteins and 19-20% of the calories demand of around half of the world's population (Shiferaw et al., 2013). Along with carbohydrates, proteins, dietary fiber, and micronutrients, wheat also contains minerals, vitamins, and fats (Sarwar et al., 2013). Grain quality consists of various characteristics that together are responsible for determining the end use of grains (Rondanini, Borrás & Savin, 2013). The chemical composition of grain i.e., starch content is considered important for ethanol production whereas protein and carbohydrate content for food and feed production. The major component of wheat endosperm is starch and it also consists of glucose polymers, amylase, amylopectin, and a small amount of protein and lipids (Fasahat, Rahman & Ratnam, 2015). The wheat grains milled into flour is used as raw material for different foods such as chapatti, bread, cakes, biscuits, muffins, pasta, noodles, sweets, and pastries (Uthayakumaran & Wrigley, 2017). The utilization of wheat grain in different forms mainly depends on protein content and gluten strength.
In bread wheat (Triticum aestivum), protein content varies from~9-13% (Žilić et al., 2011;Mohan & Gupta, 2013;Punia, Sandhu & Siroha, 2019). Wheat protein is comprised of four fractions (Osborne, 1907) which include albumins (water soluble), globulins (salt soluble), gliadins (alcohol soluble), and glutenins (diluted acid or base soluble). The 25% fraction of total wheat protein is contributed by albumin and globulin components (Belderok et al., 2000). The remaining two protein fractions, gliadins, and glutenins are collectively called gluten and share 75% of total protein and provide visco-elastic properties to the wheat dough. Therefore, the proportions of these four components are responsible for dough making and baking quality of flour (Vigni et al., 2013). Sedimentation value provides an idea about the content and quality of gluten as well as loaf volume (Pasha et al., 2007) and thus is helpful in the classification of wheat in different categories for their utilization. Grain hardness is influenced by protein content and moisture content (Turnbull & Rahman, 2002) and causes notable effects on milling as well as baking quality (Bettge, Morris & Greenblatt, 1995). Many critical factors influence the quality of wheat end-product hence it is important to assess parameters for global trade. Based on grain hardness, wheat is grouped into two categories i.e., soft wheat and hard wheat (Pomeranz & Williams, 1990).
Free radicals and reactive oxygen species (ROS) cause harmful effects on the cell membrane, enzymes, DNA, and maintenance of DNA. Oxidative damage to DNA and enzymes can result in some serious health issues such as cardiovascular diseases, carcinogenesis, type II diabetes, and obesity. Free radicals have been linked to over 60 kinds of diseases. This includes cancer, Alzheimer's disease, Parkinson's disease, and cataracts. Enzymatic antioxidants such as glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) (Narwal et al., 2014) directly react with ROS (Lobo et al., 2010) and consequently prevents oxidative damage resulting in reduced risk of oxidative-stress related chronic diseases. Enzymatic antioxidants help in the breakdown of free radicals by seeking out oxidative products and converting them into hydrogen peroxide, and then into the water. In addition, they also act as metal chelators and terminate the oxidative enzyme inhibitors hence ROS reactions (Karadag, Ozcelik & Saner, 2009). Hence the improved concentration of antioxidants helps in disease prevention & health promotion. Further, regarding mineral content, cereals and their products contribute approximately 50% of Fe 2+ and 20% of Zn 2+ in the Western diet (Velu et al., 2017). In case of wheat, Fe 2+ and Zn 2+ content was reported to be low and that too was situated in the outer layers of grains. Presently around three billion people are malnourished due to the non-availability of micronutrients as well as vitamins. A less diverse diet leads to deficiency of minerals, among which Fe 2+ and Zn 2+ deficiency causes more pronounced effects due to involvement in various metabolic processes. Micronutrient enrichment of major food crops through breeding is one of the sustainable ways to reduce malnutrition among higher-risk groups (resource-poor women, infants, and children) (Šramková, Gregová & Šturdík, 2009). It has been proven by various researchers that in the wheat genome traits related to micro-nutrient enrichment are available that could facilitate significant improvement in vitamins and minerals as well as growth-promoting components without any adverse effect on yield (Rose et al., 2001;Šramková, Gregová & Šturdík, 2009). During the course of the domestication and selection process, desirable allelic combinations were selected which led to the evolution of modern crops that feed the world today but at the cost of reduced variation resulting in the narrowing down of the genetic base of crop species (Van Heerwaarden et al., 2011) and is referred as domestication bottleneck (Hammer, 1984). For the development of high-yielding wheat varieties with improved quality for targeting specific products, several breeding programs have been initiated. As a consequence of this, wheat cultivation is based on genetically similar cultivars which resulted in a compromised genetic base due to dependency on the limited number of parents (Shuaib et al., 2010).
Wheat-related species are considered potential sources for grain yield and quality traits for which these are not fully explored. Therefore, an urgent need was sought for the characterization of these species with the aim of identification of promising lines for improved quality traits. Among ancient wheat, Triticum sphaerococcum (AABBDD, 2n = 6x = 42) is a landrace of wheat and is known as the Indian dwarf wheat. It has several advantageous features such as short and strong culms, hemispherical grains, higher protein content compared to bread wheat, and resistance to biotic and abiotic stresses, however, sphaerococcum wheat is poorly studied (Josekutty, 2008, Matsuoka, 2011. Being a hexaploid species, it may be easily exploited for bread wheat improvement to achieve food and nutritional security to fulfill United Nations' sustainable development goal. Therefore, the main objectives of this study were (1) to evaluate 34 accessions of T. sphaerococcum for important quality traits and identification of superior line for individual traits and (2) diversity assessment of T. sphaerococcum accessions based on quality traits.

MATERIALS AND METHODS
The experimental material for the present investigation comprised a diverse set of thirty-four accessions of T. sphaerococcum conserved in the national genebank namely  IC384530, IC397363, IC533826, IC53387, IC534021, IC534522, IC212160, IC534882,  IC420038, EC313761, EC541164, EC576654, EC613055, EC613057, EC187172, EC187182, EC187181, EC180062, EC187183, EC187167, EC182959, EC182945, EC10494, EC10492, EC182958, EC180041, EC180061, EC180067, EC182947, EC180066, EC182956, EC180063, EC187176, IC634028. The grain harvest was obtained from the field trial grown during Rabi 2019-20 at ICAR-National Bureau of Plant Genetic Resources (NBPGR), Issapur farm (7650′E longitude and 2840′N latitude, 223 m MSL). The soil of the farm was sandy loam in texture having organic carbon 0.7%, available N 297.6 kg/ha, available P 26.7 kg/ha, and available K 196.0 kg/ha with soil pH of 6.5. Recommended agronomic practices, irrigation, and fertilizers (120 kg N; 60 kg P 2 O 5 and 40 kg K 2 O ha −1 ) were followed throughout the experiments. Complete doses of K 2 O and P 2 O 5 were applied at the time of sowing. Nitrogen was supplied in three split doses, 60 kg N ha −1 at sowing, 30 kg N ha −1 at crown root initiation, and 30 kg N ha −1 at booting stages. The experiment was adequately irrigated with five irrigations during the crop growth. Weeding was done manually. The spray of fungicide, tilt was done to avoid the infection of rust diseases.
These accessions were evaluated biochemically following a completely randomized design with three replications for seventeen quality parameters namely thousand-grain weight (TGW), hectolitre weight (HW), sedimentation value (Sed), grain hardness index (HI), protein content (Pro), albumin content (Alb), globulin content (Glo), gliadin content (Gli), glutenin content (Glu), gluten content, lysine content (Lys), Fe 2+ and Zn 2+ content, peroxidase activity (POX), catalase activity (CAT), glutathione reductase activity (GR) and superoxide dismutase activity (SOD). The grain material was sun-dried. Grain moisture content and TGW were recorded before the biochemical estimation. For HW and HI whole grain was analyzed whereas, for the rest of the traits, flours were utilized. The flour of seeds of each accession was prepared by grinding these seeds in a cyclotec laboratory flour mill and was tested in three replicates. HW was estimated by using hectolitre apparatus. The sodium dodecyl sulphate (SDS Solution) sedimentation volume of each sample was estimated by the method suggested by Axford, McDermott & Redman (1978). HI and moisture content was analyzed using the single-kernel characterization system (SKCS) model 4100 (Perten Instruments North America, Inc., Reno, NV). Lysine was calculated by the dye-binding method as suggested by Udy (1956). Total protein content was estimated on a wet basis following Micro-Kjeldahl Method (Kjeldahl, 1883). Following the sequential Osborne extraction procedure (Osborne, 1907) the proportion of Alb, Glo, Gli, and Glu was extracted as described by Mughal et al. (2020). As gluten is composed of Gli and Glu therefore it was derived by summing up of estimation of these two contributing components. Fe 2+ and Zn 2+ content was estimated by wet digestion (15 ml acid mixture (nine concentrated nitric acid: two sulfuric acid: one perchloric acid + 0.5 g wheat flour) followed by atomic absorption spectroscopy (Jackson, 1973). Quantification of extracted proteins (Pro, Alb, Glo, Gli, and Glu) was conducted using a spectrophotometer, and BSA (Bovine Serum Albumin) was used as a standard. The enzymatic antioxidants were estimated by using the methodology suggested by Todorova et al. (2021). Eight promising accessions for grain protein content were validated by growing consecutively at NBPGR Pusa Farm and NBPGR, Issapur Farm, New Delhi during the year 2019-2020, 2020-2021, and 2021-2022.

Statistical analysis
All data were subjected to analysis of variance (ANOVA) using completely randomized design (CRD) and a comparison of treatment means was performed using Duncan's multiple range test (DMRT) at P < 0.05 using IBM SPSS Statistics software. The mean values of individual genotypes for each trait were subsequently used for analyzing summary statistics and graphs using MS Excel software. Histogram and correlation among traits were analyzed by using IBM SPSS Statistics software. Hierarchical cluster analysis was performed using Euclidean distance measure following Unweighted Pair Group Method with Arithmetic Averages (UPGMA) method by using PAleontological STatistics (PAST) software (Hammer, Harper & Ryan, 2001). The flow of work done is depicted in Fig. S1.

Total protein content (Pro), protein fraction and lysine content (Lys) estimation
Accessions significantly differed from each other for protein content and ranged from 15.34-19.35% with an average of 17.62% (Figs. S2E and 2). Maximum protein content, 19.35% was reported in accession, EC613057 followed by 19.24% in EC10494. The proportion of four protein fractions among 34 accessions of T. sphaerococcum is shown in Fig. 2. The concentration of Alb varied from 17.60 mg/g to 40.31 mg/g with the mean of 23.91 mg/g (Fig. S2F). Only one, IC534522, exhibited a moderate concentration of Alb (40.32 mg/g) whereas rest consisted of low concentration of Alb ranging from 17.60 mg/g to 30.00 mg/g ( Table 5). As far as Glo is concerned, 18 accessions showed moderate concentration and it ranged from 14.00-16.56 mg/g with the highest concentration of Glo (16.56 mg/g) in accession EC182945 (Fig. S2G). For Gli content, all the accessions belonged to low category with the range of 26.35-44.94 mg/g (Fig. S2H). In contrast, all accessions were grouped in high Glu content class with the mean of 31.00 mg/g and ranged from 24.47-39.56 mg/g (Fig. S2I). Gli and Glu are collectively called gluten which shares 85% of total protein (Fisher & Halton, 1936) and is responsible for visco-elastic property to the wheat dough. By summing up these two fractions of protein, we got a rough estimate of gluten content. With an average of 64.97 mg/g proportion of gluten among accessions, it varied from 55.33-75.06 mg/g (Fig. S2J). Significant positive correlation of gluten with Gli (r = +0.73 ÃÃ ) and Glu (r = +0.60 ÃÃ ) depicted collective contribution of these components in gluten estimation. Regarding lysine content, T. sphaerococcum wheat had an average of 0.15% of sample Lys with a range of 0.04% (EC187172) to 0.29% (EC10492) (Fig. S2K). We also observed non-significant and negative correlation (r = −0.03) between lysine and protein whereas significant negative (−0.35 Ã ) association was found with gluten (Table 4).
Adhikari et al. (2023), PeerJ, DOI 10.7717/peerj.15334 12/24 involvement in the reduction of dough adhesiveness. The CAT, GR and SOD activity varied from 0.02-0.60 unit/ml, 0.11-2.26 unit/ml, and 0.14-0.97 unit/ml respectively (Figs. S2O-S2Q). Correlation analysis results indicated that only GR and POX showed a positive correlation with HI. Whereas negative relation was observed between POX and Pro, GR and HW, CAT with Sed and Alb, and SOD with Zn 2+ content.

Cluster analysis
Thirty-four T. sphaerococcum accessions were subjected to cluster analysis by employing an unweighted pair group method with arithmetic average (UPGMA) and Euclidean distance measure. Euclidean distance matrix depicts the extent of genetic dissimilarity among accessions. The maximum distance of 86.22 was observed between EC613055 which belonged to cluster B and IC384530 which is clustered independently in cluster D from the rest of the accessions. After that EC187172 and IC384530 with 75.97 euclidean distances were grouped in clusters B and D respectively. EC187183 and IC420038 were most similar due to the lowest euclidean distance of 6.57 and both of them belonged to cluster C. Following this, pair of EC613055/EC576654 clustered in group B with a distance of 7.02. The clustering pattern of Indian dwarf wheat accessions is based on quality data and is presented in Fig. 5 and Table 6. Based on quality parameters, at a Euclidean distance of 30, 34 accessions of Indian dwarf wheat were grouped into four main clusters A, B, C, and D. Further main cluster A, B, and C is divided into two sub-clusters each whereas only one accession was grouped in main cluster D. The distribution pattern revealed the maximum number of genotypes i.e., 17 in cluster C followed by cluster B having 10 genotypes. The cophenetic correlation coefficient was estimated as 0.86 for checking the agreement between phenograms and dissimilarity matrices for the accuracy of the dendrogram. The mean performance of different clusters for the studied quality traits was shown in Table 6. Minimum mean Sed (29.45 ml) was observed among accessions of cluster B. In addition to this, cluster B was also characterized by low Gli (31.40 mg/g), Glu (30.04 mg/g), gluten content (61.44 mg/g), and high HI (90.24). Cluster B also showed a high mean value for POX (0.26 unit/ml) and GR (0.78 unit/ml) activity and hence could be targeted for wheat nutritional quality improvement. For flour yield improvement accessions clustered in cluster C could be targeted due to the maximum mean HW (81.82 kg/hl). Accessions grouped in cluster C also showed high mean values Alb (24.96 mg/g) and Lys (0.19%), whereas cluster D was characterized by high Glo (14.17 mg/g). In addition to high Glo accession of cluster D also showed high Sed (32.00 ml), Gli (41.78 mg/g), gluten (72.68 mg/g), Fe 2+ (90.71 ppm), and SOD activity (0.63 unit/ml). Cluster A demonstrated high Glu (33.26 mg/g), Zn 2+ (22.56 ppm) content, CAT (0.16 unit/ml), and GR (0.78 unit/ml) activity. To understand and confirm the dynamics of the sphaerococcum accessions, we have further carried out principal coordinate analysis (PCoA). Based on the pairwise Euclidean distance matrix, the clear distinction could be visualized among these accessions, which was in concordance with the results of the biplot (Fig. 6).

Validation of high grain protein accessions
Eight sphaerococcum accessions with high grain protein content (GPC) were validated under multi-location and multi-year trials by growing consecutively at NBPGR, Pusa Farm and NBPGR, Issapur Farm, New Delhi during the year 2019-2020, 2020-2021, and 2021-2022. Their comparative performance along with the overall mean is presented in Fig. 7. Top performing accessions with more than 15% GPC were confirmed as EC182958 (17.16%), EC187176, and EC182945 (16.16%), EC613057 (15.79%), IC634028 (15.72%) and IC533826 (15.01%) which can be used for introgression purpose and bread wheat quality improvement.

DISCUSSION
Wild relatives and ancient wheat serve as the most important genetic resources for numerous valuable traits and possess wider variation for quality traits (Zeibig, Killian & Frei, 2021). In comparison to bread wheat (T. aestivum), emmer wheat (T. dicoccoides) is reported to possess more lysine and isoleucine content (Nevo & Beiles, 1992). Similarly, as appose to cultivated wheat, and wild relatives; primitive cultivars are considered better resources for zinc and iron fortifications (Cakmak, 2008;Velu et al., 2011;Xu et al., 2011). Extensive characterization of genetic resources is a preliminary step for designing any quality breeding program. The ancient wheat, T. sphaerococcum, endemic to north-western India and southern Pakistan was withdrawn from cultivation during the early 20th century due to the introduction of the modern wheat cultivar as the result of the green revolution (Mori et al., 2013). Indian dwarf wheat is the least exploited hexaploid species for quality assessment. Here we explored T. sphaerococcum germplasm for their nutritional quality traits to enhance their potential utilization for bread wheat improvement.
In our study, a nearly similar range of HW was observed in T. sphaerococcum as reported by Mohan & Gupta (2013) in bread wheat cultivars. Higher HW means better milling quality in terms of flour yield (Martin et al., 2021) and thus accessions coming under the category of higher HW may result in better flour yield. As, HW is affected by grain shape, size, and density, therefore the possible reason for the higher value is their round grain shape. Sed recorded among these accessions was much lower than Indian bread wheat varieties (35-60 ml, (Mohan & Gupta, 2013)) and (33-52 ml, (Panghal, Chhikara & Khatkar, 2017)). Whereas, regarding the hardness index, most of the studied accessions belonged to the hard category. However, a relatively wide range (29-94) for HI in Indian bread wheat varieties was reported by Mohan & Gupta (2013). It has been observed that wheat with high protein tends to be hard whereas soft grain with low protein.
Wheat constitutes approximately 80% of the total cereal intake, and it contributes 50% of total energy and 60% of the total protein intake. Based on our findings, sphaerococcum wheat grain accumulated more protein in contrast to the narrow range (9.40-13.50) of protein in Indian bread wheat varieties reported by Mohan & Gupta (2013). We have also confirmed the high GPC accessions by growing in different environments. However, Ciaffi et al. (1992) found a large variation in T. dicoccoides, ranging from 16.00-27.00%. Similarly, in related species of wheat, mean Pro was reported as 16.67% (Jiang et al., 2008) confirming the finding that wild and primitive wheat have a wider range of variation for grain protein than cultivated ones. The Alb and Glo contribute 15-20% share of wheat flour protein. These non-prolamin protein fractions are considered nutritionally better due to a higher proportion of lysine and methionine as compared to other proteins in wheat (Lasztity, 1986). In addition to this, these proteins are also considered important for germinating and protecting embryos from insects and pathogens (DuPont & Altenbach, 2003). In our study, we have identified one accession for moderate Alb and eighteen for moderate Glo. These accessions were considered good from the nutritional point of view and could be utilized in wheat quality improvement. The Gli and Glu ratio in the present study ranged from 0.80 to 1.50 which is lower than as observed in the case of common wheat (1.60-3.80) (Geisslitz et al., 2019). It has been proven that a low Gli:Glu ratio is linked with good baking quality (Thanhaeuser, Wieser & Koehler, 2015). Therefore, these accessions are also considered good for baking purposes. Cereal-based diet and food items are poor concerning essential amino acids such as lysine and methionine (Smriga et al., 2004). Inadequate intake of Lys results in a syndrome called 'protein energy malnutrition' which is suspected to be more prevalent in developing countries. Manipulation of cereal proteins and amino acid profiles will cause a significant impact on the life of hundreds of millions of poor people. Despite extensive research aimed at the identification of Lys-rich wheat accessions; the high Lys mutants have not been discovered yet. Accessions with higher Lys estimates (EC10492, EC187176, EC182945, IC534522, and EC313761) found in our study could be utilized in wheat quality improvement.
The iron content in T. sphaerococcum accession was much higher than those obtained by Zhao et al. (2009), Mohan & Gupta (2013), Goel et al. (2018) and Khokhar et al. (2020). However, variation for Zn content is less as appose to previous findings (Goel et al., 2018;Khokhar et al., 2020) whereas in agreement with the findings of Zhao et al. (2009). It has been observed that grains with high Pro accumulate more micronutrients, which may be due to the co-localization of Pro, Zn 2+ , and Fe 2+ in the embryo and the aleurone layer of the grain (Khokhar et al., 2020). Zn 2+ concentration showed a non-significant weak correlation with TGW (0.22) which is in close agreement with the findings of Khokhar et al. (2020). A significant positive association (+0.43 Ã ) between Fe 2+ and Zn 2+ contents indicated the possibility of simultaneous improvement of both micronutrients (Goel et al., 2018). It was also stipulated that there is the involvement of a common mechanism for the accumulation of both micronutrients (Hernandez-Espinosa et al., 2020). Likewise, a significant positive association between Fe 2+ and Zn 2+ was observed by various researchers (Velu et al., 2017;Hernandez-Espinosa et al., 2020;Khokhar et al., 2020). Further, the probable reason for the association between Fe 2+ and Zn 2+ content was found to be the co-localization of quantitative trait loci present at chromosome 3B governing these traits (Velu et al., 2017). Due to the lack of ample genetic variation in cross-compatible species, the traditional breeding for micronutrient enhancement was non-exploitable. Henceforth hexaploid species T. sphaerococcum could be exploited in this direction. For antioxidants, maximum CAT, GR, and SOD activity were recorded in the case of accessions EC18294, EC180063, and EC182956 respectively. CAT was reported to be involved in oxidative reaction during bread making and its highest activity was recorded during the kernel development stage and was responsible for the detoxification of H 2 O 2 (Kruger & Leberg, 1974). Likewise, SOD protects the oxidation of cellular components through reactive oxygen species (Mughal et al., 2020). Hence accessions showing high activity for antioxidants could be targeted for wheat quality improvement in terms of nutrition. GR may control the ratio of GSSG (oxidized glutathione)/GSH (reduced glutathione) in wheat flour and play a role in determining the physical properties of wheat flour doughs (Every et al., 2006).
Based on association analysis, there were non-significant positive estimates of the correlation coefficient between Sed and Pro; however, it was significantly correlated as per the findings of Drikvand et al. (2013). Further, a significant positive correlation was observed between Pro and HI which is supported by the finding of Drikvand et al. (2013). HI and TGW were negatively associated at 0.05% level of significance, likewise non-significant but negative correlation estimates were observed by Abdipour et al. (2016). Also, it has been proven that Lys is negatively correlated with Pro as reported by Rharrabti et al. (2001).
The clustering pattern based on quality parameters revealed 17 accessions in cluster C followed by cluster B having 10 genotypes with cophenetic correlation coefficient value as 0.86 greater than the acceptable level of 0.85 (Stuessy, 2009). Minimum mean Sed was observed among accessions of cluster B. In addition to this, cluster B was also characterized by low Gli, Glu, gluten content, and high HI. Cluster B also showed a high mean value for POX and GR activity, hence, could be targeted for wheat nutritional quality improvement. For flour yield improvement, accessions clustered in cluster C could be targeted due to the maximum mean HW. Accessions grouped in cluster C also showed a high mean value of Alb and Lys, whereas cluster D was characterized by high Glo. As Alb and Glo protein is reported nutritionally better due to a higher proportion of lysine and methionine (Lasztity, 1986) therefore these accessions could be utilized in common wheat improvement for essential amino acids. In addition to high Glo, accession of cluster D also showed high Sed, Gli, gluten, Fe 2+ , and SOD activity. Also, accession of cluster D could be targeted for enhancement of protein content in common wheat as they are characterized by high protein content. Cluster A demonstrated high Glu, Zn 2+ content, CAT, and GR activity henceforth could be exploited in the improvement of respective traits.

Future implications
Based on the comprehensive estimation of grain quality parameters, our study revealed that the primitive wheat landrace, Indian dwarf wheat T. sphaerococcum is a rich source of grain protein content, lysine content, iron and zinc content, and antioxidant activity. It also has low gluten content which is a major dietary requirement based on consumer preference. Therefore exploration, conservation, and use of collections belonging to this species may be promoted to gain maximum benefit for the well-being of mankind. Among the studied accessions, EC10492, and EC313761 (from USSR) were found superior for a maximum of seven traits followed by EC180063 and EC182958 for five traits. We also validated high GPC accessions such as EC182958 (from Canada), EC187176, EC182945, EC613057 (from France), IC634028 (Kathod Gehun from Tapi district of Gujarat), and IC533826 with more than 15% grain protein content.

CONCLUSIONS
Wild relatives, ancient species, and landraces are rich valuable genetic resources for abiotic and biotic stress tolerance, and nutritional qualities and play a major role in wheat improvement. In the present study, we analyzed 34 T. sphaerococcum, Indian dwarf wheat accessions conserved in the national genebank of India for seventeen-grain quality parameters. We found sufficient variation among accessions for all studied quality traits and accessions with superior quality traits were identified. Among these accessions, EC10492, and EC313761 were found superior for a maximum of seven traits followed by EC180063 and EC182958 for five traits. We also validated high GPC accessions and EC182958 (17.16%), EC187176 and EC182945 (16.16%), EC613057 (15.79%), IC634028 (15.72%) and IC533826 (15.01%) were confirmed with more than 15% GPC. The superior accessions identified in the present study could be the potential genetic resource for a breeder to improve the wheat's nutritional quality. The sphaerococcum wheat should be promoted for cultivation on the farmers' field considering its richness in grain nutrients and end-use quality with low gluten, high protein, high iron, and zinc content, and high antioxidant activity. Further, the germplasm explorer should prioritize the collection of this species from unexplored areas to harness its nutritional richness for alleviating nutritional imbalance and improving the health of mankind. The results of the present study showed the possibility of utilization of Indian dwarf wheat in wheat quality breeding aiming for nutritional security under the United Nation's sustainable development goals (SDG).